Strategies to edit paralogous genes with CRISPR/Cas9



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Abstract

The purpose of this study is to develop the strategies of CRISPR/Cas9 application to improve fidelity and specificity of this platform. Here we use a model system, which includes target gene and a paralogue - potential aim for off-target double-strand break induction. The study was carried on using Brattleboro rats embryonic fibroblasts which are homozygous for a mutation in arginine-vasopressin gene (target). The potential off-target gene is oxytocin gene: its DNA sequence is almost identical to that of arginine-vasopressin gene. To prevent off-target effect we designed several strategies, which were further used on Brattleboro rats embryonic fibroblasts. Here we show, that these strategies allowed us to generate double-strand breaks in arginine-vasopressin gene without any off-target effects in oxytocin gene. The endonuclease restriction assay shows that we have modified arginine-vasopressin gene while using both CRISPR/Cas9 and single-stranded oligonucleotides as a donor for homologous recombination. At last, if we consider Brattleboro rats as a model of monogenic disease the strategies designed could be translated in human therapeutic genome editing studies.

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About the authors

A. A Nemudryi

Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation

T. B Malankhanova

Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation; National Research University Novosibirsk State University

A. A Malakhova

Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation

S. P Medvedev

Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation

S. M Zakian

Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation

Email: zakian@bionet.nsc.ru

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